Zebrafish (Danio rerio) - Ulm...• up to 40 mm in length (snout to base of caudal fin), up to 700...

Zebrafish (Danio rerio)as model system

Prof. Dr. Gilbert WeidingerInstitute of Biochemistry and Molecular Biology

Ulm University

Zebrafish taxonomy

Taxon Euteleostomi (bonyvertebrates, like human)

Superclass Osteichthyes (bony fish). This includes the tetrapods!

Class Actinopterygii (ray-finned fish, „Strahlenflosser“), tetrapods belongto the clade of lobe-finned fish(Sarcopterygii „Fleischflosser“)

Subclass Teleostei („echte Knochenfische“)

Order Cypriniformes(„Karpfenfische“)

there are 44 species in the genusDanio

Betancur-R, Ricardo; et al. (2013). "The Tree of Life and a New Classification of Bony Fishes". PLOS Currents Tree of Life (Edition 1). doi:10.1371/currents.tol.53ba26640df0ccaee75bb165c8c26288.

Habitat

freshwater fish

indigenous to Indian subcontinent(India, Bangladesh, Nepal, Myanmar, Pakistan)

„Danio“ is derived from Bengali wordfor „in the rice field“

prefer shallow, slow-flowing or still water (floodplains, ponds, rice fields)

in the wild found at 16.5 to 33 °C, slightly alkaline pH (7.9-8.2)

occopy the whole of the water column

equally likely in open water and in vegetation

form shoals

life-span in the wild unclear

Spence et al. Biol. Rev. (2008), 83, pp. 13–34

Gilbert Weidinger, BIOTECGroup leader Sonderforschungsbereich 655

Features

• up to 40 mm in length (snout to base of caudal fin), up to 700 mg• up to 3 years of age in the lab• mouth pointing upwards, lower jaw protruding further than upper• 5 to 7 dark stripes

• 2 pairs of fins: pectoral and pelvic• 3 unpaired fins: dorsal, anal, caudal

Sexual dimorphism

females:

prominent belly (eggs)interstripe color whitishyellow dorsal fin

males:

slender body shapeinterstripe color pinkish (most obvious at anal fin)white dorsal fin

Imaging of early development

Imaging of early development

In toto imaging

Transparency of early embryos

Transparency

Design of ENU forward genetic screens

Patton and Zon (2001).

Nat Rev Gen, 2, 956

Examples of mutants found in forward screens

Patton and Zon (2001).

Nat Rev Gen, 2, 956

Examples for screens

Patton and Zon (2001).

Nat Rev Gen, 2, 956

Examples for screens

Patton and Zon (2001).

Nat Rev Gen, 2, 956

Transgenesis

Kawakami et al. (2004),

Dev Cell 7, 133-144.

Transgenesis

Kawakami et al. (2004),

Dev Cell 7, 133-144.

Transgenesis

The tol2 transposon system

Kawakami (2007)

Genome Biology 8, S7

The Tol2 transposon system

BAC transgenesis

Suster et al. BMC

Genomics 2009, 10:477

Transgenesis in zebrafish

G0„founder“

F1

F1

F2

mosaic, chimeric

T/+ „hemizygous“

T/+ „hemizygous“

microinject plasmid + Tol2 capped sense RNA

Zebrafish as model system

representative of its animal group good

relevance for humans good

many progeny excellent

progeny all year round excellent

fast generation time OK

easy to house/culture good

small (but not too small) good

cheap good

fast embryonic development excellent

external development excellent

transparency (imaging!) excellent

accessible for embryological techniques (transplantations) good

diploid (or haploid): ability to identify mutants good

small genome OK

inbred lines poor

forward genetics excellent

reverse genetics excellent

RNAi NO

transgenesis excellent

pluripotent stem cell culture (ES cells) NO

knock-ins/outs in development

Housing in the lab

re-circulating freshwater systems (5-10% water exchange per day)

reverse osmosis water (RO) + defined amount of saltsOR: mixture of tap water (in Germany!) + RO water + salts

Water parameters

Parameter Represents Target Comments Controlled by

Conductivity Total ionconcentration

200 – 1000 µSWeidinger: 350µS (Siemens)

Higher might help to reduceenergetic cost (osmoticbalance); but not good to usesalts that contain a lot of NaCl

Addition of CaCO3 & MgCO3 + trace elements

And/or „Red Sea Salt“, „Instant Ocean“ or the like

Total hardness(GH)

All multivalent ions, particularCa2+ and Mg2+

5 ° dH Too little causes bone andother defects

Carbonate (temporary) hardness (KH)

Bicarbonate(HCO3

-)3 ° dH Too much causes limescale

Copper 0 Is toxic No use of copper pipes!

Phosphate (PO4) < 5 mg/l Fish don‘t care much, but high concentrations favor algaegrowth

Amount and type of food.

pH 7 - 8Weidinger: 7.3

Sodium bicarbonate (usuallypH needs to be brought up)

Temperature 24 – 30 °C (28.5°C)Weidinger: 27°C

Compromise between fast growth and bearable climate in room.

Air temperature needs to besimilar to minimizeevaporation.

Oxygen At saturation:7.8 mg/l at 28°C

Water flow, recirculation ofwater in storage tank

Nitrogen cycle

N excreted by fish (urine, feces) ends up in the water. Needs to bedealt with. Bacteria in the filtersmetabolize it.

Nitrogen cycle

Formula English Deutsch relevance Target Comment

NH3 Ammonia Ammoniak Very toxic Cannot be measured Should not accumulate, since it converts to NH4+

But: at high pH it might accumulate!

NH4+ Ammonium Ammonium harmless < 0.02 mg/l Harmless, but should not accumulate if bacteria-

mediated nitrogen cycle works

NO2 Nitrite Nitrit Toxic < 10 mg/l

NO3 Nitrate Nitrat Rel. harmless

< 50 mg/l Remove by water exchange

Disease: Mycobacterium

Whipps et al. (2012), ILAR 53, 85-105.

Disease: microsporidiosis

Sanders et al. (2012), ILAR Journal 52, 106.

intracellular parasites

most prominent: Pseudoloma neurophilia

infects muscle, central nervous system, ovaries

reduced growth, emanciation, spinal deformation

often sub-clinical

detected in 75% of all fish facilities

Sanders et al. (2012), ILAR Journal 52, 106.

gram-positive spores in follicles and stroma of ovary

Hygiene

Recirculating water is filtered and sterilized

debris is allowed to settle in sump water is coarsely filtered through filter mats (which also contain

bacteria) water is fine filtered in pressurized filters water is UV sterilized

Hygiene

Cleanliness

1. feces and left-over food is removed from bottom oftanks

2. tank surfaces and lids are kept clean

3. removable tanks are washed (dishwasher) regularily

4. NO plants (real or artificial) are used

5. snails can be used to manage algae

Health management

Monitoring

• fish status is monitored 2x daily

• dead or sick fish are removed (euthanised) immediately

• sick fish can be submitted to pathology services (ZIRC, Oregon)

Precautions

• stress is kept at a minimum (water parameters, nutrition, density)

• fish are never imported into main facility > quarantene > only embryos are transfered

• old fish are euthanized (> 2 years)

• embryos are bleached to remove parasites from chorions (which does not help against microsporidiosis)

• no street shoes and/or disinfection mats

Stocking density

Zebrafish are social, form shoals > don‘t like to be kept individually for long periodsof time

Zebrafish diplay social order, in particular males. 2 males kept together will fight > avoid that, rather keep individually or in larger groups.

• Fish well-being as measured by reproductive success is not adversely affected byhigh stocking density (12 fish per liter).Weidinger: Usually 5/liter, permit to use 7/liter.

Castranova et al., Zebrafish 2011.

Food

In the wild: omnivorous. Zooplankton, insects (mostly aquatic, but also from surface), phytoplankton, algae, plant material...

Fish dry food flakes (eg. Tetramin)must be refrigerated and administered dry.

Live food: artemia brine shrimp.purchased as cysts. Hatch within 48h in aerated high salt water.

Adults: 1-3 times a day. Can easily survive for 7 days without food.

Breeding

Carlos Manuel Díaz

• day-night cycle (12-14 h day, 10-12 h night)

• fish spawn in the morning (till noon)• male + female must spend the night

together• rel. small space (1 l per pair)• they eat their progeny!

variations:• timed egg lay via separation of male +

female• mass-spawning: eg. 4 males + 4 females• in vitro fertilization

Larval rearing

Embryos/larvae survive on yolk for 5 days.

Day 5 – 12: Continuous food supply is best,no or very slow water flow.

Different foods based on size of larvae:paramecia (easily cultured protozoa) OR rotifers (Protostomia)dry foods of increasing grain size, as often as possible

Growth rate dependsheavily on nutrition andstocking density. (Weidinger: 10 larvae/liter)

Sexual maturity can bereached within 5 weeks, usually within 2.5 months.

Resources

Zebrafish International Resource Center (ZIRC), University of Oregon

protocols for husbandry, pathology services, source for wild-type and transgenic / mutant fish lines

European Zebrafish Resource Center, Karlsruhe Institute for Technology, ezrc.kit.edu

European repository for fish lines, screening facility

Zebrafish model organism database (ZFIN). zfin.org

Info on fish lines (transgenic, mutant), research reagents (antibodies, morpholinos), genomeannotation

Zebrafish husbandry organisation. zhaonline.org

Non-profit, promotes husbandry standards through education & research

European Society for Fish Models in Biology and Medicine (EuFishBioMed)

promotes collaboration and exchange between fish labs